Reciprocal epithelial-mesenchymal interactions are the key step in early tooth formation and defects in these interactions are likely to lead to tooth agenesis. Bone morphogenetic protein 4 (Bmp4), a crucial signal molecule in development of many tissues, is very likely required at early stages during tooth formation. Its spatial and temporal expression pattern correlates highly with the epithelial-mesenchymal interactions during initiation of tooth formation. Local application of recombinant Bmp4 to tooth buds in vitro can mimic the action of the dental epithelium during tooth induction. However, there is not direct in vivo evidence to show the importance of Bmp4 during tooth formation, since Bmp4 knockout mice die at an early stage of embryonic development prior to tooth development. Msx1 knockout mice have shown an early arrest of tooth formation, which is associated with substantial reduction of Bmp4 in arrested tooth germs. Addition of recombinant Bmp4 to Msx1-arrested tooth germs in vitro permits further tooth development (bud to cap stage). Previously, the applicants cloned the mouse Bmp4 gene and characterized its promoter. Msx homeobox binding sites were identified in the Bmp4 promoter. Msx expression plasmids up-regulate Bmp4 promoter activity in in vitro transfection assays. Using transgenic mice harboring lacZ gene driven by various Bmp4 promoter fragments, tooth- specific elements of BMp4 expression were narrowed down to the region between -1144 to -260 bp. Seven transgenic mouse lines with various expression levels of human Bmp4 cDNA have been generated. The aim of this proposal is to investigate the role of Bmp4 and its regulation by Msx1 during normal and abnormal tooth formation in vivo. The underlying hypothesis for the proposed studies is that Msx1 acts upstream of Bmp4 at the transcriptional level and that Bmp4 is required for tooth formation. This will be tested by using transgenic mice and Msx1 knockout mice. Specific Aim 1 will examine the expression changes of a luciferase or lacZ gene, driven by intact or specific Bmp4 promoter fragments, in the presence and absence of Msx1 to determine whether Msx1 can activate the Bmp4 in vivo. Specific Aim 2 will examine whether overexpression of Bmp4 cDNA, driven by specific Msx1 promoter or the Bmp4 promoter fragments, can rescue the tooth formation defect in Msx1 knock out mice in vivo. This bypass experiment will address the regulatory roles of Msx1 on Bmp4, and examine roles of Bmp4 during tooth formation in vivo. Specific Aim 3 will extend these animal experiments to examine potential mutations of the Msx1 gene in autosomal dominant forms of familial tooth agenesis. Research into this relatively unexplored area will contribute to our understanding of the basic Msx1- Bmp4 signal pathway, mechanisms of tooth formation, and possible cause of autosomal dominant forms of familial tooth agenesis.